Modulators and modulation of the interaction between RGM and Neogenin

ABSTRACT

This invention relates to drug screening using mammalian repulsive guidance molecules and mammalian Neogenin. In addition, the invention provides for methods of preventing, alleviating or treating various disorders of the nervous system, angiogenic disorders or disorders of the cardio-vascular system and malignancies of different etiology by disrupting the interaction between RGM and Neogenin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 USC §371 toPCT/US03/20147, filed Jun. 26, 2003, which claims the benefit of U.S.application Ser. No. 60/392,062, filed Jun. 26, 2002.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the field of neuroscience and neurology. Inparticular embodiments it is related to the area of axon guidance cuesand their modulators, and neurological drug screening using repulsiveguidance molecules and Neogenin.

BACKGROUND OF THE INVENTION

One of the most important mechanisms in formation of embryonic nervoussystems is the guidance of axons and growth cones by directionalguidance cues (Goodman, Annu. Rev. Neurosci. 19 (1996), 341-77; Mueller,Annu. Rev. Neurosci 22, (1999), 351-88). A suitable model system forstudying this guidance process is the retinotectal system ofvertebrates. In the chick embryo approximately 2 million retinalganglion cell (RGC) axons leave each eye and grow towards thecontralateral tectum opticum to form a precise map (Mey & Thanos,(1992); J. Hirnforschung 33,673-702). Having arrived at the anteriorpole of the optic tectum, RGC axons start to invade their tectal targetto find their target neurons. Mapping occurs in such a way that RGCaxons from nasal retina project to posterior tectum and temporal axonsto anterior tectum. Along the dorso-ventral axis, axons coming fromdorsal retina terminate in ventral tectum, whereas those from ventralretina end up in dorsal tectum. Ultimately, a precise topographic map isformed, where neighborhood relationships in the retina are preserved inthe tectum so that axons from neighboring ganglion cells in the retinasynapse with neighboring tectal neurons. Most important for formation ofthis map are graded tectal guidance cues, read by retinal growth conescarrying corresponding receptors which also show a graded distribution(Sperry, Proc. Natl. Acad. Sci. USA 50 (1963), 703710; Bonhoeffer &Gierer, Trends Neurosci. 7 (1984) 378-381).

Position of each retinal growth cone in the tectal field is thereforedetermined by two sets of gradients: receptor gradients on in-growingretinal axons and growth cones and ligand gradients on tectal cells(Gierer, Development 101 (1987), 479-489). The existence of the gradedtectal ligands has been postulated from anatomical work. Theiridentification, however, proved to be extremely difficult and was onlymade possible with the development of simple in vitro systems (Walter ;Development 101 (1987), 685-96; Cox, Neuron 4 (1990), 31-7). In thestripe assay, RGC axons grow on a membrane carpet, consisting ofalternating lanes of anterior (a) and posterior (p) tectal membranes. Onthese carpets, temporal retinal axons grow on anterior tectal membranesand are repelled by the posterior lanes, whereas nasal axons do notdistinguish between a and p membranes (Walter, Development 101 (1987),685-96). The same specificity is also observed in the growth conecollapse assay (Raper & Kapfhammer, Neuron 4 (1990), 21-29) wheretemporal retinal growth cones collapse after addition of posteriortectal membrane vesicles but do not react to anterior tectal vesiclesand where nasal growth cones are insensitive to either type of vesicles(Cox, (1990), loc. cit.). In both assay systems, treatment of posteriortectal membranes with the enzyme phosphatidylinositol-specificphospholipase C (PI-PLC) (which cleaves the lipid anchor ofglycosylphosphatidylinositol (GPI)-linked proteins) removed theirrepellent and collapse-inducing activity (Walter, J. Physiol 84 (1990),104-10).

One of the first repulsive guidance molecules identified in theretinotectal system of chick embryos was a GPI-anchored glycoproteinwith a molecular weight of 33/35 kDa (Stahl, Neuron 5 (1990), 735-43).This 33/35 kDa molecule, later termed RGM (Repulsive Guidance Molecule),was active in both stripe and collapse-assays and was shown to beexpressed in a low-anterior high-posterior gradient in the embryonictecta of chick and rat (Mueller, Curr. Biol. 6 (1996), 1497-502;Mueller, Japan Scientific Societies Press (1997), 215-229). Due to theabnormal biochemical behavior of RGM, the precise amino acid sequencewas not easily obtainable. RGM was described as a molecule which isactive during vertebrate development. Interestingly, RGM isdownregulated in the embryonic chick tectum after E12 and in theembryonic rat tectum after P2 and completely disappears after theembryonic stages (Muller (1992), Ph. D thesis University of Tübbingen;Müller (1997) Japan Scientific Societies, 215-229). In 1996, Müller(loc. cit.) showed that CALI (chromophore-assisted laser inactivation)of RGM eliminates the repulsive guidance activity of posterior tectalmembranes. However, due to the presence of other guidance molecules, inparticular of RAGS (repulsive axon guidance signal) and ELF-1 (Ephligand family 1), a complete elimination of guidance was not alwaysdetected and it was speculated that RGM acts in concert with RAGS (nowtermed ephrin-A5) and ELF-1 (ephrin-A2). It was furthermore envisagedthat RGM may be a co-factor potentiating the activity of RAGS and ELF-1in embryonic guidance events.

In 1980/81 the group of Aguayo found that, when peripheral neurons aretransplanted/grafted into injured CNS of adult, axon growth of CNSneurons is induced (David, Science 214 (1981), 931-933). Therefore, itwas speculated that CNS neurons have still the ability and capacity ofneurite-outgrowth and/or regeneration, if a suitable environment wouldbe provided. Furthermore, it was speculated that “CNS-neuronregeneration inhibitors” may exist.

In 1988, Caroni and Schwab (Neuron 1,85-96) described two inhibitors of35 kDa and 250 kDa, isolated from rat CNS myelin (NI-35 and NI-250; seealso Schnell, Nature 343 (1990) 269-272; Caroni, J. Cell Biol. 106(1988), 1291-1288).

In 2000, the DNA encoding for NI-220/250 was deduced and thecorresponding potent inhibitor of neurite growth was termed Nogo-A(Chen, Nature 403 (2000), 434-438). The membrane-bound Nogo turned outto be a member of the reticulon family (GrandPre, Nature 403 (2000),439-444).

Further factors which mediate neuronal outgrowth inhibition have firstbeen isolated in grasshoppers, and termed “fasciclin IV” and later“collapsin” in chicken. These inhibitors belong to the so-calledsemaphorin family. Semaphorins have been reported in a wide range ofspecies and described as transmembrane proteins (see, inter alia,Kolodkin Cell 75 (1993) 1389-99, Püschel, Neuron 14 (1995), 941-948).Yet, it was also shown that not all semaphorins have inhibitoryactivity. Some members of the family, e.g. semaphorin E, act as anattractive guidance signal for cortical axons (Bagnard, Development 125(1998), 5043-5053).

A further system of repulsive guidance molecules is the ephrin-Ephsystem. Ephrins are ligands of the Eph receptor kinases and areimplicated as positional labels that may guide the development of neuraltopographic maps Flanagan, Ann. Rev. Neurosc. 21 (1998), 309-345).Ephrins are grouped in two classes, the A-ephrins which are linked tothe membrane by a glycosylphosphatidylinositol-anchor (GPIanchor) andthe B-ephrins carrying a transmembrane domain (Eph nomenclaturecommittee 1997). Two members of the A-ephrins, ephrin-A2 and ephrin-A5,expressed in low anterior-high posterior gradients in the optic tectum,have recently been shown to be involved in repulsive guidance of retinalganglion cell axons in vitro and in vivo (see, inter alia (Drescher,Cell 82 (1995), 359-70; Cheng, Cell 79 (1994), 157-168; Feldheim, Neuron21 (1998), 563-74; Feldheim, Neuron 25 (2000), 563-74). Considering thefact that a plurality of physiological disorders or injuries are relatedto altered cellular migration processes, the technical problemsunderlying the present invention was to provide for means and methodsfor modifying developmental or cellular (migration) processes which leadto disease conditions.

The Ephrin, Semaphorin, Slit, and RGM families of extracellular guidancecues specify axonal trajectories during nervous system development¹⁻³.The netrins are a family of proteins that are profound modulators ofgrowth of developing axons, functioning as attractants for some axonsand repellents of other axons. As such, the modulation of these effectsprovides an important therapeutic pathway for assisting the regenerationof axons in adult nervous system (e.g. following injury or trauma).While neuronal receptors have been identified for most axonal guidancecues, the mechanism by which the recently sequenced RGM protein (WO02/051438) acts has not been clarified³. As described in part above,chick RGM is expressed in a posterior to anterior tectal gradient andhas been shown to collapse temporal but not nasal retinal growth cones³.After signal peptide cleavage and GPI addition, the cell surface RGM isproteolytically processed to a mature active form of 33 kDa³.

The ability to construct high-throughput and specific pharmaceuticalscreens for modulators of guidance cues (such as RGM) has been limitedby the lack of identifiable receptors. Identifying receptors on axonsthat mediate neural responsiveness to guidance cues will provide keytargets for identifying lead pharmaceuticals for therapeuticintervention in the nervous system (see, for example, U.S. Pat. Nos.6,087,326 and 5,747,262). Accordingly, because RGM has a demonstratedrole in axon growth, it would be desirable to accurately identify thereceptor through which RGM acts such that targeted screens could beconducted.

Neogenin is known to share sequence similarity with the Netrin receptorDeleted in Colorectal Cancer (DCC). The sequence for the Neogenin genehas been described (for example, Keeling S L, Gad J M, Cooper H M.“Mouse Neogenin, a DCC-like molecule, has four splice variants and isexpressed widely in the adult mouse and during embryogenesis.” Oncogene.Aug. 7, 1997;15(6):691-700. GenBank NT_(—)039474; NM_(—)008684) and ithas been previously theorized that it is an interaction with Netrin-1that is responsible for signaling through Neogenin. However, asdescribed in detail herein, the present inventors have determined thetrue physiological ligand for Neogenin.

SUMMARY OF THE INVENTION

The invention identifies Neogenin as the receptor for Repulsive GuidanceMolecule. Accordingly, the invention envisions the use of the previouslydescribed Neogenin and RGM molecules in combinations and methods whichcould not previously have been suggested. In particular, the inventionallows for targeted screening assays and the development of inhibitorscapable of specifically inhibiting the interaction between RGM andNeogenin.

The invention provides efficient methods of identifying agents,compounds or lead compounds for agents capable of modulating Neogenincellular function. Generally, these screening methods involve assayingfor compounds which modulate mammalian Neogenin interaction with anatural mammalian RGM. A wide variety of assays for binding agents areprovided including labeled in vitro protein-protein binding assays,immunoassays, cell based assays, animal based assay, etc. Preferredmethods are amenable to automated, cost-effective high throughputscreening of chemical libraries for lead compounds. Such librariesencompass candidate agents of numerous chemical classes, thoughtypically they are organic compounds; preferably small organic compoundsand are obtained from a wide variety of sources including libraries ofsynthetic or natural compounds. Identified agents find use in thepharmaceutical industries for animal and human trials; for example, theagents may be derivatized and rescreened in in vitro and in vivo assaysto optimize activity and minimize toxicity for pharmaceuticaldevelopment.

In vitro binding assays employ a mixture of components includingmammalian Neogenin protein, which may be part of a fusion product withanother peptide or polypeptide, e.g. a tag for detection or anchoring,etc. The assay mixtures comprise a natural extracellular mammalianNeogenin binding target, such as a RGM. While native binding targets maybe used, it is frequently preferred to use portions (e.g. peptides)thereof so long as the portion provides binding affinity and avidity tothe subject mammalian Neogenin protein conveniently measurable in theassay. The assay mixture also comprises a candidate pharmacologicalagent and typically, a variety of other reagents such as salts, buffers,neutral proteins, e.g. albumin, detergents, protease inhibitors,nuclease inhibitors, antimicrobial agents, etc. The mixture componentscan be added in any order that provides for the requisite bindings andincubations may be performed at any temperature which facilitatesoptimal binding. The mixture is then incubated under conditions whereby,but for the presence of the candidate pharmacological agent, themammalian Neogenin protein specifically binds the cellular bindingtarget, portion or analog with a reference binding affinity. Incubationperiods are likewise selected for optimal binding but also minimized tofacilitate rapid, high-throughput screening.

After incubation, the agent-biased binding between the mammalianNeogenin protein and one or more binding targets is detected. Aseparation step is often initially used to separate bound from unboundcomponents. Separation may be effected by precipitation (e.g. TCAprecipitation, immunoprecipitation, etc.), immobilization (e.g on asolid substrate), etc., followed by washing by, for examples, membranefiltration, gel chromatography (e.g. gel filtration, affinity, etc.).One of the components usually comprises or is coupled to a label. Thelabel may provide for direct detection such as radioactivity,luminescence, optical or electron density, etc. or indirect detectionsuch as an epitope tag, an enzyme, etc. A variety of methods may be usedto detect the label depending on the nature of the label and other assaycomponents, e.g. through optical or electron density, radiativeemissions, nonradiative energy transfers, etc. or indirectly detectedwith antibody conjugates, etc. A difference in the binding affinity ofthe mammalian Neogenin protein to the target in the absence of the agentas compared with the binding affinity in the presence of the agentindicates that the agent modulates the binding of the mammalian Neogeninprotein to the mammalian RGM. Analogously, in a cell-based transcriptionassay, a difference in the mammalian Neogenin transcriptional inductionin the presence and absence of an agent indicates the agent modulatesvertebrate such induced transcription. A difference, as used herein, isstatistically significant and preferably represents at least a 50%, morepreferably at least a 90% difference.

The invention provides methods and compositions for identifyingpharmacological agents useful in the diagnosis or treatment ofneurological disease or injury. In particular, the invention providesmixtures comprising an isolated (RGM) and an isolated Neogenin receptorcapable of specifically binding said RGM. The general methods involveincubating a mixture comprising an isolated RGM, an isolated Neogeninreceptor, and a candidate pharmacological agent, and determining if thepresence of the agent modulates the binding of the RGM to the receptor.Specific agents provide lead compounds for pharmacological agents usefulin the diagnosis or treatment of neurological disease or injury.

It is an object of the present invention to provide a method ofmonitoring the interaction of a RGM and a Neogenin.

Another object of the invention is to provide a method for monitoringthe interaction between RGM and Neogenin so that agonists andantagonists can be identified.

Another object of the invention is to provide a polypeptide useful forantagonizing the interaction between a RGM and a Neogenin receptor.

Another object of the invention is to provide a polypeptide useful forantagonizing the interaction between RGM and Neogenin.

These and other objects of the invention are achieved by one or more ofthe embodiments described below. In one embodiment a method ofmonitoring the interaction of a RGM and a Neogenin receptor is provided.The method comprises the steps of:

-   -   contacting a first protein comprising an RGM with a second        protein which comprises Neogenin under conditions where a domain        of the RGM binds to a domain of the Neogenin;    -   determining the binding of the first protein to the second        protein or second protein to the first protein.

According to another aspect of the invention a method is provided formonitoring the interaction between a RGM and a Neogenin. The methodcomprises the steps of:

-   -   contacting a fusion protein comprising an RGM domain with cells        which express a Neogenin;    -   detecting the fusion protein comprising the RGM domain which        binds to the cells.

As another aspect of the invention a method is provided for monitoringthe interaction between a RGM and Neogenin. The method comprises thesteps of:

-   -   contacting a protein comprising a RGM domain with cells which        express a polypeptide comprising Neogenin;    -   detecting the protein comprising the RGM domain which binds to        the cells.

As still another aspect of the invention a polypeptide portion ofNeogenin useful for antagonizing the interaction between RGM andNeogenin is provided.

According to still another aspect of the invention a method ofmonitoring the interaction between a RGM and Neogenin is provided. Themethod comprises the steps of:

-   -   co-culturing in a matrix (a) embryonic nerve cells with (b)        cells which have been transfected with an expression construct        encoding a RGM and which express the Neogenin;    -   adding to the cells an inhibitor of binding of the RGM and        Neogenin;

determining the axon outgrowth adjacent to the cells which express theRGM in the presence and absence of inhibitor.

As another aspect of the invention a method is provided for monitoringthe interaction between a RGM and Neogenin. The method comprises thesteps of:

-   -   culturing embryonic nerve cells under conditions in which they        display growth cones;    -   contacting the embryonic nerve cells with a RGM and an        anti-Neogenin antibody;    -   observing the effect of the antibody on the collapse of the        growth cones.

Yet another aspect of the invention is provided by an antibodypreparation which specifically binds to a Neogenin protein.

Yet another aspect of the invention is provided by an antibodyspecifically targeted to domain(s) involved in the interaction betweenNeogenin and RGM. In a particular embodiment, such antibodies would bedirected towards the FNIII domain(s) of Neogenin.

Yet another aspect of the invention is provided by an antibodypreparation which specifically binds to a RGM protein.

RGM is processed proteolytically, and the active domain extends carboxylfrom the cut site to the GPI anchorage site. This same region appear tomediate Neogenin binding. Accordingly, antibodies directed to domainswithin this region would be one aspect of the invention.

Yet another aspect of the invention is provided by a nucleic acidcapable of inhibiting the expression of an RGM protein or a Neogeninprotein.

The medical applications of such compounds, their agonists, and theirantagonists are enormous and include preventing, alleviating or treatingvarious disorders of the nervous system, angiogenic disorders ordisorders of the cardio-vascular system and malignancies of differentetiology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Neogenin is a high affinity binding site for RGM.

(A) Purified recombinant RGM-AP is avoided by temporal axons in a stripeassay.

(B) The binding of a chick RGM-AP to COS-7 cells expressing mouseNeogenin is illustrated. The bound protein is detected as a darkreaction product on the right.

(C) Saturation of RGM-AP to COS-7 expressing Neogenin. Bound RGM-APactivity was determined for each of the indicated concentrations ofRGM-AP. The data are the average ±sem of 6 independent determinations.

(D) Scatchard analysis of RGM-AP binding to Neogenin expressing cells.Data from (C) are replotted. The Kd is 230 pM.

(E′, E″ and E′″) Chick-RGM-A-AP, Mouse RGM-A-AP and RGM-B-AP also bindto Neogenin.

FIG. 2. Physical complex containing RGM and Neogenin.

(A) Co-immunoprecipitation of RGM and Neogenin. HEK293T cells weretransfected with plasmids encoding the indicated proteins andimmunoprecipitated with anti-Myc antibody resin. The presence ofNeogenin protein in the lysates and the immunoprecipitate isillustrated.

(B) RGM affinity chromatography of adult mouse brain. Adult mouse brainmembrane fractions were extracted with 2% TritonX-100 and solubilizedprotein was incubated with or without chick RGM-AP-Myc-His. Proteinretained by an anti-Myc antibody resin was analyzed by anti-Neogeninimmunoblot.

FIG. 3. Specificity of Neogenin interaction with RGM.

(A) RGM-AP binding to COS-7 cells transfected with expression plasmidsencoding various Netrin-1 binding proteins.

(B) RGM-AP binds to the FNIII repeats of Neogenin. RGM-AP binding toCOS-7 expressing the indicated fragments of Neogenin is illustrated.

(C) Netrin-1 does not alter the binding of RGM-AP to Neogenin. Theability of chick RGM-AP to bind to COS-7 cells expressing wild typemouse Neogenin was assessed in the presence or absence of conditionedmedium containing Netrin-1-Myc (100 nM).

FIG. 4. Dominant negative Neogenin-1 blocks retinotectal axonalpreferences.

(A) Retinal ganglion cell axon preference for anterior tectal membranes.Outgrowth from explants from the temporal or nasal chick retina (green)is illustrated on stripes consisting of anterior (black) or posterior(red) tectal membranes.

(B) Retinotectal stripe assay in the presence of soluble ectodomain ofNeogenin-1. A preference of temporal axons for anterior membranesobserved in (A) is greatly reduced.

(C) Stripe preference as a function of soluble Neogenin ectodomainconcentration. Data from 4-16 experiments such as in (A) and (B) werescored for the extend of preference of temporal retinal axons forAnterior versus Posterior stripes (AP) or Anterior versus Anteriorstripes (AA). A rating of 2 reflects a complete segregation of axons tothe anterior stripes, a rating of 1 reflects still a preference foranterior stripes without complete segregation and 0 is nopreference^(10,11).

(D) Binding specificity of the RGM/Neogenin and ephrinA/EphA pairs.COS-7 cells were transfected with expression plasmids for Neogenin-1 orEphA3 and then incubated with medium containing Ephrin-A2-Fc,Ephrin-A5-Fc or RGM-AP. Note the specificity of binding. Bound Fc hasdetected with HRP-conjugated anti-human IgG.

FIG. 5. Amino acid sequence of Neogenin (SEQ ID NO: 1).

FIG. 6. Nucleotide sequence of Neogenin (SEQ ID NO: 2).

(A) Nucleotides 1-3480.

(B) Nucleotides 3481-5199.

DETAILED DESCRIPTION

To search for high affinity RGM binding sites in brain, a fusion proteinof chick RGM truncated amino terminal to the GPI site was fused to humanplacental alkaline phosphatase (AP) to express a soluble,carboxy-terminally MycHis tagged secreted protein. When expressed inHEK293 cells, the cRGM-AP fusion protein was processed proteolyticallyto yield a 110 kDa fusion protein (data not shown). This materialretained biological activity as demonstrated by the avoidance of cRGM-APstripes by temporal retinal ganglion cell axons (FIG. 1A).

The cRGM-AP fusion protein does not bind to COS-7 kidney-derived cells,so we expressed an adult mouse brain cDNA expression library in thesecells and screened for clones driving expression of cell surface bindingactivity. Only a single clone was identified in screens of 480,000independent clones. This cDNA clone expressed a saturable binding sitefor cRGM-AP with a Kd of 230 pM (FIG. 1B-D). DNA sequence analysisrevealed that the high affinity RGM-AP binding protein was Neogenin. Inthe mouse genome there are three RGM-related sequences, that we havetermed mRGM-A, mRGM-B and mRGM-C. The three mouse RGM sequences share41-49% aa identity and 55-61% aa similarity with one another. Chick RGMshares the highest level of identity with mRGM-A at 80% aa identity and84% aa similarity. Both RGM-A and RGM-B are expressed in many regions ofthe developing mouse brain, so we tested if AP fusion proteins of thesealso bind to Neogenin. Both mRGM-A and mRGM-B bind mNeogenin with highaffinity (FIG. 1E).

To verify that the RGM interaction with Neogenin was due to theirparticipation in a physical complex, RGM and Neogenin were co-expressedin HEK293T cells and analyzed by co-immunoprecipitation. RGMprecipitates contained detectable Neogenin but control immunoprcipitatesdid not (FIG. 2A). More relevant for in vivo activity, affinitychromatography using the RGM-AP protein isolated Neogenin protein fromadult mouse brain tissue (FIG. 2B).

The RGM interaction with Neogenin raises several issues of specificitysince Neogenin has been described previously as a Netrin bindingprotein⁴. RGM and Netrin-1 show no significant sequence similarity³. Itshould be noted that the reported Netrin binding affinity to Neogenin(˜2nM) is an order of magnitude less than the RGM affinity (230pM). Weconsidered whether RGM-AP binds to other known Netrin receptors. DCC ismost closely related to Neogenin in sequence and the two proteins arereported to have similar affinities for Netrins. Functional studies havedemonstrated a role for DCC in mediating axonal guidance byNetrins^(6,7), but the role of Neogenin in mediating axonal responses toNetrins have not been documented. DCC has no detectable affinity forRGM-AP, any affinity must be at least 50 fold less than for Neogenin(FIG. 3A). Unc5 proteins can bind Netrin-1 independently of DCC, andserve as obligate co-receptors together with DCC in axon repulsion byNetrins⁸. However, neither Unc5H1 nor Unc5H3 proteins bind RGM-AP (FIG.3A). Netrin-1 is known to bind to the FNIII region of DCC, rather thanthe Ig domains⁹. Similarly, full RGM binding affinity is obtained with atruncated Neogenin containing only the FNIII repeats (FIG. 3B). Thisraises the possibility that RGM and Netrin bind to similar regions ofNeogenin. However, the addition of excess Netrin-1 did not appear toalter RGM-AP binding to Neogenin (FIG. 3C). While functionalinteractions between Netrin and RGM might exist, the two protein do notappear to compete for binding to a single site on Neogenin.

To assess the role of Neogenin binding in RGM signalling in retinalganglion axons, we purified the soluble recombinant ectodomain ofNeogenin via a His tag, and tested its function-blocking capability. Tothe extent that RGM-Neogenin signalling contributes to retinotectaltargeting, the soluble Neogenin ectodomain should disrupt the temporalretinal preference for anterior versus posterior tectal membranes in astripe assay. The preference of temporal but not nasal retinal ganglioncell axons is obvious in the stripe assay under control conditions (FIG.4A, C), as reported previously^(10,11). In the presence of solubleNeogenin the preference of temporal axons for anterior tectal stripes islost (FIG. 4B). Blockade of retinal axon stripe preference isdose-dependent, with essentially complete blockade at 400 nM solubleNeogenin ectodomain (FIG. 4C). The tectum contains ananterior-to-posterior both RGM and ephrin A2/A5 guidance cues^(3,5). Toensure that soluble Neogenin ectodomain was selectively blocking RGMfunction, and not ephrin function, we tested the ligand bindingspecificity of these systems (FIG. 4D). It is clear that ephrinA2/5 bindto EphA3 but not Neogenin and that RGM binds to Neogenin but not EphA3.Thus, the blockade of stripe preference by soluble Neogenin demonstratesa crucial role for RGM/Neogenin signalling in determining retinotectalaxon guidance in vitro.

RGMs form high affinity complexes with Neogenin and Neogenin/RGMcomplexes play a significant role in retinotectal guidance systems.Since the Neogenin-related DCC functions as an axonal guidance receptorfor Netrin-1, there is precedence for this family of receptor proteinsmediating axonal guidance in vivo^(6,7). The interaction of RGM withNeogenin is of higher affinity than Netrin-1's interaction withNeogenin, and is specific amongst Netrin-binding proteins. ThusNeogenin's primary role in nervous system development is as a RGMreceptor, with DCC serving as the primary Netrin receptor. It is ofinterest that both Neogenin and RGM (data not shown) are highlyexpressed in adult nervous system and in the injured nervous system,thus implicating them in adult neural regeneration. For example, RGM islocalized at very high concentrations in brains at the lesion site inhumans suffering form traumatic brain injury or from cerebral ischemiaFrom approximately 1-7 days post injury or post cerebral ischemia,monocytes, lymphocytes, granulocytes and a few neurons express RGM. Insubsequent stages, RGM is present on fibroblast-like cells, on reactiveastrocytes and in fresh and mature scar tissue which forms at the lesionsite.

The invention provides methods and compositions for identifyingpharmacological agents useful in the diagnosis or treatment ofmammalian, particularly human, neurological disease or injury. Themethods rely on monitoring the interaction of a mammalian, particularlyhuman, RGM and a corresponding Neogenin in the presence and absence of acandidate agent. A wide variety of assays can be used, includingreceptor activation assays and binding assays. Binding assays maymonitor RGM binding to a domain of or a full-length receptor expressedon a cell, or in vitro protein-protein binding of a RGM to a full lengthor truncated receptor. In some embodiments, such in vitro screensinvolve the immobilization of one of the binding partners on a solidsubstrate.

Typically, these assays involve a mixture comprising an isolated RGM andan isolated Neogenin capable of specifically binding said RGM. We havedemonstrated that these mammalian gene products function as naturalmammalian, and in particular, human, ligand-receptor complex. Thegeneral methods comprise the steps of: (1) forming a mixture comprisingan isolated RGM, an isolated negoenin-1 receptor, and a candidatepharmacological agent; (2) incubating said mixture under conditionswhereby, but for the presence of said candidate pharmacological agent,said RGM specifically binds said Neogenin at a first binding affinity;and. (3) detecting a second binding affinity of said RGM to saidNeogenin, wherein a difference between said first and second bindingaffinity indicates that said candidate pharmacological agent is a leadcompound for a pharmacological agent useful in the diagnosis ortreatment of neurological disease or injury.

The term “modulator” as employed herein relates to “inhibitors” as wellas “activators” of RGM or Neogenin function. Most preferably said“modulation” is an inhibition, wherein said inhibition may be a partialor a complete inhibition. An inhibitor of RGM, for example, need notbind RGM but might inhibit RGM by interacting with Neogenin andinhibiting the RGM/Neogenin interaction. In addition, the inhibitorcould inhibit RGM by inhibiting transcription, translation or processing(pre or post-translational) of RGM. Similarly, a modulator may mimic RGMfunction through binding to Neogenin without sharing homology to RGM.

The term, RGM amino acid sequence relates to the RGM polypeptidesdisclosed in WO 02/051438 (to which the following SEQ ID Nos. refer). Inparticular, SEQ ID NOs: 20 and 21 depict human RGM1. Human RGM1 has beenlocalized on chromosome 15. Further, human RGMs comprise RGM2 and RGM3.RGM2 is depicted in SEQ ID NO: 23 (amino acid sequence) and is encodedby a nucleotide sequence as shown in SEQ ID NO: 22. Human RGM2 has beenlocalized on chromosome 5. Furthermore, human RGM3 is shown in appendedSEQ ID NO: 25 (amino acid sequence) and encoded by a nucleotide sequenceas depicted in SEQ ID NO: 24. Human RGM3 is located on chromosome 1.Yet, as will be discussed herein below, said term relates also tofurther RGM homologues.

The term “(poly) peptide” means, in accordance with the presentinvention, a peptide, a protein, or a (poly) peptide which encompassesamino acid chains of a given length, wherein the amino acid residues arelinked by covalent peptide bonds. However, peptidomimetics of such RGMproteins/(poly) peptides or Neogenin proteins/(poly)peptides whereinamino acid (s) and/or peptide bond (s) have been replaced by functionalanalogs are also encompassed by the invention.

The present invention is not restricted to uses of RGM and Neogenin fromhuman, mouse or chicken and its inhibitors but also relates to the useof inhibitors of RGM and Neogenin or of RGM and Neogenin itself (orfunctional fragments or derivatives thereof) from other species. Sincethe present invention provides for the use of amino acidsequences/polypeptides of RGM and Neogenin and their correspondinginhibitors and since the amino acid sequences of human and chicken RGMand Neogenin have been disclosed, the person skilled in the art isprovided with the information to obtain RGM and Neogenin sequences fromother species, like, inter alia, mouse, rat, pig, etc. The relevantmethods are known in the art and may be carried out by standard methods,employing, inter alia, degenerate and non degenerate primers inPCR-techniques.

Basic molecular biology methods are well known in the art and, e.g.,described in Sambrook (Molecular Cloning; A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)) and Ausubel, “Current Protocols in Molecular Biology”, GreenPublishing Associates; and Wiley Interscience, N.Y. (1989).

Furthermore, as employed in the context of the present invention, theterms “RGM”, “RGM modulator”, “RGM-inhibitor”, “Neogenin”, “Neogeninmodulator” and “Neogenin inhibitor” also relate to RGM and Neogeninmolecules (and their corresponding inhibitors) which are variants orhomologs of the RGM and Neogenin molecules (and their inhibitors) asdescribed herein. “Homology” in this context is understood to refer inthis context to a sequence identity of RGM and Neogenin of at least 70%,preferably more than 80% and still more preferably more than 90% on theamino acid level. The present invention, however, comprises also(poly)peptides deviating from wildtype amino acid sequences of human orchicken RGM and Neogenin, wherein said deviation may be, for example,the result of amino acid and/or nucleotide substitution(s), deletion(s),addition(s), insertion(s), duplication(s), inversion(s) and/orrecombination(s) either alone or in combination. Those deviations maynaturally occur or be produced via recombinant DNA techniques well knownin the art. The term “variation” as employed herein also comprises“allelic variants”. These allelic variations may be naturally occurringallelic variants, splice variants as well as synthetically produced orgenetically engineered variants.

FIGS. 5 and 6 present the amino acid sequence and nucleotide sequence,respectively, for Neogenin. For the purposes of this application,reference to “wildtype” Neogenin refers to this sequence although, asdescribed herein, significant modifications of this sequence will notdepart from the spirit of the invention. To the extent that allelic orother differences occur among Neogenin genes, these differences may beused to create specific probes or antibodies.

The term “polynucleotide” in accordance with the present inventioncomprises coding and, wherever applicable, non-coding sequences (likepromoters, enhancers etc.). It comprises DNA, RNA as well as PNA. Inaccordance with the present invention, the term “polynucleotide/nucleicacid molecule” comprises also any feasible derivative of a nucleic acidto which a nucleic acid probe may hybridize. Said nucleic acid probeitself may be a derivative of a nucleic acid molecule capable ofhybridizing to said nucleic acid molecule or said derivative thereof.The term “nucleic acid molecule” further comprises peptide nucleic acids(PNAs) containing DNA analogs with amide backbone linkages (Nielsen,Science 254 (1991), 1497-1500). The term “nucleic acid molecule” whichencodes a RGM (poly) peptide or a functional fragment/derivativethereof, in connection with the present invention, is defined either by(a) the specific nucleic acid sequences encoding said (poly) peptidespecified in the present invention or (b) by nucleic acid sequenceshybridizing under stringent conditions to the complementary strand ofthe nucleotide sequences of (a) and encoding a (poly) peptide deviatingfrom the nucleic acid of (a) by one or more nucleotide substitutions,deletions, additions or inversions and wherein the nucleotide sequenceshows at least 70%, more preferably at least 80% identity with thenucleotide sequence of said encoded RGM and Neogenin (poly)peptideshaving an amino acid sequence as defined herein and functions as a RGMor Neogenin (or a functional fragment/derivative thereof) as the casemay be.

The term “modulator” as employed herein also comprises the term“inhibitor”, as mentioned herein above. The term comprises “modulators”of the RGM and Neogenin polypeptides and/or the RGM and Neogeninencoding nucleic acid molecule/genes. In context of this invention itis-also envisaged that said “modulation” may lead, when desired, to anactivation of RGM and/or Neogenin.

The term “functional fragment or derivative thereof” in context of thepresent invention and in relation to the RGM and Neogenin moleculescomprises fragments of the RGM and Neogenin molecules defined hereinhaving a length of at least 10, in another embodiment 25, in anotherembodiment at least 50, in another embodiment at least 75, and inanother embodiment at least 100 amino acids depending on the applicationas would be known to one of skill in the art.

Functional fragments of the herein identified RGM and Neogenin moleculesor RGM and Neogenin molecules of other species (homologous RGM andNeogenin) may be comprised in fusion and/or chimeric proteins.“Functional fragments” comprise RGM or Neogenin fragments (or theirencoding nucleic acid molecules) which are able to replace RGM orNeogenin full length molecules in corresponding assays (as disclosed in,e. g. collapse and/or stripe assays) or may elucidate an anti-RGM oranti-Neogenin specific immune-response and/or lead to specific anti-RGMor anti-Neogenin antibodies. An example of such a “functional fragment”would be a fragment of RGM capable of binding Neogenin. In context ofthe present invention, polynucleotides encoding functional fragments ofRGM or Neogenin and/or their derivatives have at least 15, in anotherembodiment at least 30, in another embodiment at least 90, in anotherembodiment at least 150, and in another embodiment at least 300nucleotides depending on the application as would be known to one ofskill in the art.

The term “derivative” means in context of their invention derivatives ofRGM and Neogenin molecules and/or their encoding nucleic acid moleculesand refer to natural derivatives (like allelic variants) as well asrecombinantly produced derivatives/variants which may differ from RGM orNeogenin molecules by at least one modification/mutation, e. g. at leastone deletion, substitution, addition, inversion or duplication. The term“derivative” also comprises chemical modifications. The term“derivative” as employed herein in context of the RGM and Neogeninmolecule also comprises soluble RGM and Neogenin molecules which do notcomprise any membrane anchorage.

As mentioned herein above, the present invention provides for the use ofa modulator, preferably an inhibitor, of RGM molecules and/or theircorresponding encoding polynucleotides/nucleic acid molecules for thepreparation of a pharmaceutical composition for preventing, alleviatingor treating various disorders of the nervous system, angiogenicdisorders or disorders of the cardio-vascular system and malignancies ofdifferent etiology.

In a preferred embodiment, said disorders of the nervous system comprisedegeneration or injury of vertebrate nervous tissue, in particularneurodegenerative diseases, nerve fiber injuries and disorders relatedto nerve fiber losses.

Said neurodegenerative diseases may be selected from the groupconsisting of motorneuronal diseases (MND), amyotrophic lateralsclerosis (ALS), Alzheimer disease, Parkinsons disease, progressivebulbar palsy, progressive muscular atrophy, HIV-related dementia andspinal muscular atrophy(ies), Down's Syndrome, Huntington's Disease,Creutzfeldt-Jacob Disease, Gerstmann-Straeussler Syndrome, kuru,Scrapie, transmissible mink encephalopathy, other unknown priondiseases, multiple system atrophy, Riley-Day familial dysautonomia saidnerve fiber injuries may be selected from the group consisting of spinalcord injury(ies), brain injuries related to raised intracranialpressure, trauma, secondary damage due to increased intracranialpressure, infection, infarction, exposure to toxic agents, malignancyand paraneoplastic syndromes and wherein said disorders related to nervefiber losses may be selected from the group consisting of paresis ofnervus facials, nervus medianus, nervus ulnaris, nervus axillaris,nervus thoracicus longus, nervus radialis and for of other peripheralnerves, and other aquired and non-aquired deseases of the (human)central and peripheral nervous system.

The above mentioned spinal cord and brain injuries not only comprisetraumatic injuries but also relate to injuries caused by stroke,ischemia and the like. It is in particular envisaged that the inhibitorsas defined herein below and comprising, inter alia, anti-RGM antibodiesbe employed in the medical art to stimulate nerve fiber growth inindividuals, in particular in vertebrates, most preferably in humans.

In a more preferred embodiment of the present invention, the inventionprovides for the use of a modulator, preferably an inhibitor to RGM (ora functional fragment or derivative thereof) for the preparation of apharmaceutical composition for the treatment of disorders of thecardio-vascular system, wherein these disorders, e. g., comprisedisorders of the blood-brain barrier, brain oedema, secondary braindamages due to increased intracranial pressure, infection, infarction,ischemia, hypoxia, hypoglycemia, exposure to toxic agents, malignancy,paraneoplastic syndromes.

It is envisaged, without being bound by theory, that RGM inhibitors maystimulate or allow surviving neurons to project collateral fibers intothe diseased tissue, e. g. the ischemic tissue.

RGM is expressed locally at the side of artificial transection ofbrain/spinal cord tissue in test animals (like rats), e. g., in thepenumbra region surrounding an ischemic core of a human suffering focalischemia in the temporal cortex. Furthermore, it is documented in thethat RGM is, surprisingly, expressed in tissue(s) affected by traumaticbrain injuries. The invention also relates to the use of a RGMpolypeptide or a functional fragment or derivative thereof or the use ofa polynucleotide encoding the same (polypeptides and polynucleotides asdefined herein), wherein the above described disease or conditionassociated with seizures is epilepsy. An epilepsy is therebycharacterized by an epileptic seizure as a convulsion or transientabnormal event experienced by the subject, e. g. a human patient, due toa paroxysmal discharge of (cerebral) neurons. The epileptic seizurescomprise tonic seizures, tonic-clonic seizures (grand mal), myoclonicseizures, absence seizures as well as akinetic seizures. Yet, alsocomprised are in context of this invention simple partial seizures, e.g. Jacksonian seizures and seizures due to perinatal trauma and/or fetalanoxia. As mentioned herein below, the uses described herein relate inparticular to the preparation of pharmaceutical compositions for thetreatment of diseases/conditions associated with aberrant sprouting ofnerve fibers, like epilepsy; see also Routbort, Neuroscience 94 (1999),755-765.

In a even more preferred embodiment of the invention, the modulator,preferably the inhibitor of RGM (or of its functional fragment orderivative thereof or of its encoding nucleic acid molecule) is used forthe preparation of a pharmaceutical composition for the modification ofneovascularization. Said modification may comprise activation as well asstimulation. It is in particular envisaged that said neovascularisationbe stimulated and/or activated in diseased tissue, like inter alia,ischemic and/or infarctious tissue. Furthermore, it is envisaged thatthe RGM-inhibitors described herein may be employed in the regulation ofthe blood-brain barrier permeability.

It is furthermore envisaged that said modulators, preferably saidinhibitors for RGM be employed in the alleviation, prevention and/orinhibition of progression of vascular plaque formation (e. g.artherosclerosis) in cardio-vascular, cerebo-vascular and/ornephrovascular diseases/disorders.

Furthermore, the present invention provides for the use of a modulator,preferably an inhibitor of RGM as defined herein for the preparation ofa pharmaceutical composition for remyelination. Therefore, the presentinvention provides for a pharmaceutical composition for the treatment ofdemyelinating diseases of the CNS, like multiple sclerosis or ofdemyelinating diseases like peripheral neuropathy caused by diphteriatoxin, Landry-Guillain-Barre-Syndrom, Elsberg-Syndrom,Charcot-Marie-Tooth disease and other polyneuropatias. A particularpreferred inhibitor of RGM in this context is an antibody directedagainst RGM, e.g. an IgM antibody. It has previously be shown thatcertain IgMs bind to oligodendrocytes and thereby induce remyelination.IgM antibodies against RGM are known in the art and comprise e.g. theF3D4 described in the appended examples.

In addition the invention provides for the use of a RGM polypeptide asdefined herein or of a functional fragment or derivative thereof or of apolynucleotide encoding said polypeptide or fragment or derivative forthe preparation of a pharmaceutical composition for preventing,alleviating or treating diseases or conditions associated with theactivity of autoreactive immune cells or with overactive inflammatorycells.

Most preferably these cells are T-cells.

Furthermore, the present invention relates to the use of a modulator,preferably an inhibitor or another RGM binding molecule of a RGMpolypeptide or of a functional fragment or derivative thereof or of apolynucleotide encoding said polypeptide or of fragment/derivativethereof for modifying and/or altering the differentiation status ofneuronal stem cells and/or their progenitors. Said stem cells arenormally found in the subventricular zones of many brain regions. It isknown that factors in the microenvironment of the brain dramaticallyinfluence the differentiation of undifferentiated stem cells. It isassumed that due to the characteristic expression of RGM in thesubventricular layers of many different brain regions, this moleculecould be a marker for stem cells. Furthermore, RGM inhibitors, likeantibodies could be useful markers for stem cells. Most important instem cell biology is the understanding of factors influencing theirdifferentiation. It is therefore assumed that RGM inhibitors change thedevelopmental fate of these cells.

RGM is not only expressed in ischemic tissue but is also expressed inscar tissue surrounding (brain) lesions.

It is particularly preferred that the modulator, preferably theinhibitor of the RGM molecule (or its functional fragment or derivative)is an antibody or a fragment or a derivative thereof, is an aptamer, isa specific receptor molecule capable of interacting with a RGMpolypeptide or with a functional fragment or derivative thereof, or is aspecific nucleic acid molecule interacting with a polynucleotideencoding an RGM and/or the polypeptide.

The antibody to be used in context of the present invention can be, forexample, polyclonal or monoclonal antibodies. Techniques for theproduction of antibodies are well known in the art and described, e. g.in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988. The production of specific anti-RGM antibodies isfurther known in the art (see, e. g. Mutter (1996) loc. cit.) ordescribed in the appended examples.

The term “antibody” as employed herein also comprises chimeric, singlechain and humanized antibodies, as well as antibody fragments, like,inter alia, Fab fragments.

Antibody fragments or derivatives further comprise F(ab′)2, Fv or scFvfragments; see, for example, Harlow and Lane, loc. cit. Variousprocedures are known in the art and may be used for the production ofsuch antibodies and/or fragments, see also appended examples. Thus, the(antibody) derivatives can be produced by peptidomimetics. Further,techniques described for the production of single chain antibodies (see,inter alia, U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to polypeptide (s) of this invention. Also, transgenicanimals may be used to express humanized antibodies to polypeptides ofthis invention. Most preferably, the antibody to be used in theinvention is a monoclonal antibody, for example the F3D4 antibodydescribed in the appended examples may be employed when an IgM isdesired. The general methodology for producing, monoclonal antibodies iswell-known and has been described in, for example, Kohler and Milstein,Nature 256 (1975), 494-496 and reviewed in J. G. R. Hurrel, ed.,“Monoclonal Hybridoma Antibodies: Techniques and Applications”, CRCPress Inc., Boco Raron, Fla. (1982), as well as that taught by L. T.Mimms et al., Virology 176 (1990), 604-619.

Preferably, said antibodies (or inhibitors) are directed againstfunctional fragments of the RGM polypeptide. As pointed out herein aboveand as documented in the appended examples, such functional fragmentsare easily deducible for the person skilled in the art and,correspondingly, relevant antibodies (or other inhibitors) may beproduced.

The “modulator”, preferably the “inhibitor” as defined herein may alsobe an aptamer.

In the context of the present invention, the term “aptamer” comprisesnucleic acids such as RNA, ssDNA (ss=single stranded), modified RNA,modified ssDNA or PNAs which bind a plurality of target sequences havinga high specificity and affinity.

Aptamers are well known in the art and, inter alia, described inFamulok, Curr. Op. Chem. Biol. 2 (1998), 320-327. The preparation ofaptamers is well known in the art and may involve, inter alia, the useof combinatorial RNA libraries to identify binding sites (Gold, Ann.Rev. Biochem. 64 (1995), 763-797). Said other receptors may, forexample, be derived from said antibody etc. by peptidomimetics.

Other specific “receptor” molecules which may function as inhibitors ofthe RGM polypeptides are also comprised in this invention. Said specificreceptors may be deduced by methods known in the art and comprisebinding assays and/or interaction assays. These may, inter alia, involveassays in the ELISA-format or FRET-format.

Said “inhibitor” may also comprise specific peptides binding to and/orinterfering with RGM.

Furthermore, the above recited “modulator”, preferably “inhibitor” mayfunction at the level of RGM gene expression. Therefore, the inhibitormay be a (specific) nucleic acid molecule interacting with apolynucleotide encoding a RGM molecule (or a functional fragment orderivative thereof.) These inhibitors may, e. g., comprise anti sensenucleic acid molecules, small inhibitory RNAs (siRNAs) or ribozymes.

The nucleic acid molecule encoding RGM or Neogenin may be employed toconstruct appropriate anti-sense oligonucleotides or siRNA molecules.

Said anti-sense oligonucleotides are able to inhibit the function ofwild-type (or mutant) RGM and Neogenin genes and comprise, for example,at least 15 nucleotides, at least 20 nucleotides, at least 30nucleotides or at least 40 nucleotides.

In addition, ribozyme approaches are also envisaged for use in thisinvention.

Ribozymes may specifically cleave the nucleic acid molecule encoding RGMor Neogenin.

In the context of the present invention ribozymes comprise, inter alia,hammerhead ribozymes, hammerhead ribozymes with altered core sequencesor deoxyribozymes (see, e. g., Santoro, Proc. Natl. Acad. Sci. USA 94(1997), 4262) and may comprise natural and in vitro selected and/orsynthesized ribozymes.

Nucleic acid molecules according to the present invention which arecomplementary to nucleic acid molecules coding for proteins/ (poly)peptides regulating, causing or contributing to obesity and/or encodinga mammalian (poly) peptide involved in the regulation of body weight(see herein below) may be used for the construction of appropriateribozymes (see, e. g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360257) which specifically cleave nucleic acid molecules of the invention.Selection of the appropriate target sites and corresponding ribozymescan be done as described for example in Steinecke, Ribozymes, Methods inCell Biology 50, Galbraith, eds. Academic Press, Inc. (1995), 449-460.

Said “inhibitor” may also comprise double-stranded RNAs, which lead toRNA mediated gene interference (see Sharp, Genes and Dev. 13 (1999),139-141). Further potential inhibitors of RGM or Neogenin may be foundand/or deduced by interaction assay and employing corresponding read-outsystems. These are known in the art and comprise, inter alia, two hybridscreenings (as, described, inter alia, in EP-0 963 376, WO 98/25947, WO00/02911), GST-pull-down columns, co-precipitation assays from cellextracts as described, inter alia, in Kasus-Jacobi, Oncogene 19 (2000),20522059, “interaction-trap” systems (as described, inter alia, in U.S.Pat. No. 6,004,746), expression cloning (e. g. lambda gt11), phagedisplay (as described, inter alia, in U.S. Pat. No. 5,541,109), in vitrobinding assays and the like. Further interaction assay methods andcorresponding read out systems are, inter alia, described in U.S. Pat.No. 5,525,490, WO 99/51741, WO 00/17221, WO 00/14271 or WO 00/05410.

In yet another embodiment, the present invention provides for the use ofthe RGM amino acid sequence or of a functional fragment or derivativethereof or of a polynucleotide encoding said polypeptide or fragment orderivative for the preparation of a pharmaceutical composition forpreventing or treating tumor growth or formation of tumor metastases.

RGM (naturally isolated or recombinantly produced) and/or functionalfragments thereof may be employed for the preparation of apharmaceutical composition for the treatment of neoplastic disorders, inparticular of disorders related to tumor (cell) migration, metastasisand/or tumor invasion. Furthermore, it is envisaged that RGM inhibitsundesired neovascularisation. Said neovascularisation, as an angiogenicdisorder during neoplastic events, should be prevented in order tolimit, inter alia, tumor growth.

Growth cones of neurons and (invasive) tumor cells secrete a cocktail ofproteases (uPA, tPA, MNPs, etc.) in order to degrade extracellularmatrix. Furthermore, similar mechanisms for adhesion and (cell)migration are employed by these cellular systems. RGM and/or itsfunctional fragments may be employed to actively stimulate withdrawal oflamellipodia of tumor cells and/or to induce their collapse.

In addition the invention provides for the use of a RGM polypeptide asdefined herein or of a functional fragment or derivative thereof or of apolynucleotide encoding said polypeptide or fragment or derivative forthe preparation of a pharmaceutical composition for preventing,alleviating or treating diseases or conditions associated with theactivity of autoreactive immune cells or with overactive inflammatorycells.

Most preferably these cells are T-cells.

In yet another embodiment, the invention provides for the use of a theRGM polypeptide h or of a functional fragment or derivative thereof orof a polynucleotide encoding said polypeptide or fragment or derivativefor the preparation of a pharmaceutical composition for the treatment ofinflammation processes and/or allergies, for wound healing or for thesuppression/alleviation of scar formation. Scar tissue is formed byinvading cells, most importantly by fibroblasts and/or glial cells.Migration and adhesion of these cells are required to get to the lesionside. RGM or an active fragment/derivative could prevent accumulation ofthese cells in the lesion side, thereby preventing or slowing down scarformation. In inflammatory reactions cells migrate to the inflamedregion and RGM or its active fragment/derivative prevent or reducemigration of these cells to the side of inflammation, thereby preventingoveractive inflammatory reactions.

In context of the present invention, the term “pharmaceuticalcomposition” also comprises optionally further comprising an acceptablecarrier and/or diluent and/or excipient. The pharmaceutical compositionof the present invention may be particularly useful in preventing and/ortreating pathological disorders in vertebrates, like humans. Saidpathological disorders comprise, but are not limited to, neurological,neurodegenerative and/or neoplastic disorders as well as disordersassociated with seizures, e. g. epilepsy. These disorders comprise,inter alia, Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis (FALS/SALS), ischemia, stroke, epilepsy, AIDS dementiaand cancer.

The pharmaceutical composition may also be used for prophylacticpurposes.

Examples of suitable pharmaceutical carriers are well known in the artand include phosphate buffered saline solutions, water, emulsions, suchas oil/water emulsions, various types of wetting agents, sterilesolutions etc. Compositions comprising such carriers can be formulatedby well known conventional methods. These pharmaceutical compositionscan be administered to the subject at a suitable dose.

Administration of the suitable compositions may be effected by differentways, e. g., by intravenous, intraperitoneal, subcutaneous,intramuscular, topical, intradermal, intranasal or intrabronchialadministration. However, it is also envisaged that the pharmaceuticalcompositions are directly applied to the nervous tissue. The dosageregimen will be determined by the attending physician and clinicalfactors. As is well known in the medical arts, dosages for any onepatient depends upon many factors, including the patient's size, bodysurface area, general health, age, sex, the particular compound to beadministered, time and route of administration, and other drugs beingadministered concurrently. Pharmaceutical active matter may be presentpreferably, inter alia, in amounts between 1 ng and 1000 mg per dose,more preferably in amounts of 1 ng to 100 mg however, doses below orabove this exemplary range are envisioned, especially considering theaforementioned factors. If the regimen is a continuous infusion, itshould also be in the range of 1 ug to 10 mg units per kilogram of bodyweight per minute, respectively. Progress can be monitored by periodicassessment. The compositions of the invention may be administeredlocally or systemically. Administration will generally be parenterally,e. g., intravenously. The compositions of the invention may also beadministered directly to the target site, e. g., by biolistic deliveryto an internal or external target site or by catheter to a site in anartery. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents, depending on the intended use ofthe pharmaceutical composition. Such agents may be drugs acting on thecentral nervous system as well as on small, unmyelinated sensory nerveterminals (like in the skin), neurons of the peripheral nervous systemof the digestive tract., etc.

It is also understood that the pharmaceutical composition as definedherein may comprise nucleic acid molecules encoding RGM and Neogenin(and/or functional fragments or derivatives thereof) or correspondingRGM and Neogenin inhibitors as defined herein. As mentionedherein-above, said inhibitors comprise, but are not limited to,antibodies, aptamer, RGM-interacting peptides as well as inhibitorsinteracting with the RGM-encoding polynucleotides.

Accordingly, the present invention also provides for a method oftreating, preventing and/or alleviating pathological disorders andconditions as defined herein, whereby said method comprisesadministering to a subject in need of such a treatment a pharmaceuticalcomposition/medicament as defined herein. Preferably, said subject is ahuman.

The nucleic acid molecules may be particularly useful in gene therapyapproaches and may comprise DNA, RNA as well as PNA. Said nucleic acidmolecules may be comprised in suitable vectors, either inter alia, geneexpression vectors. Such a vector may be, e. g., a plasmid, cosmid,virus, bacteriophage or another vector used e. g. conventionally ingenetic engineering, and may comprise further genes such as marker geneswhich allow for the selection of said vector in a suitable host cell andunder suitable conditions.

Furthermore, the vectors may, in addition to the nucleic acid sequencesencoding RGM and Neogenin or the corresponding inhibitors, compriseexpression control elements, allowing proper expression of the codingregions in suitable host cells or tissues.

Such control elements are known to the artisan and may include apromoter, translation initiation codon, translation and insertion sitefor introducing an insert into the vector. Preferably, the nucleic acidmolecule of the invention is operatively linked to said expressioncontrol sequences allowing expression in (eukaryotic) cells.Particularly preferred are in this context control sequences which allowfor correct expression in neuronal cells and/or cells derived fromnervous tissue.

Control elements ensuring expression in eukaryotic cells are well knownto those skilled in the art. As mentioned above, they usually compriseregulatory sequences ensuring initiation of transcription and optionallypoly-A signals ensuring termination of transcription and stabilizationof the transcript. Additional regulatory elements may includetranscriptional as well as translational enhancers, and/ornaturally-associated or heterologous promoter regions. Possibleregulatory elements permitting expression in for example mammalian hostcells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter(Rous sarcoma virus), human elongation factor la-promoter, CMV enhancer,CaM-kinase promoter or SV40-enhancer. For the expression for example innervous tissue and/or cells derived therefrom, several regulatorysequences are well known in the art, like the minimal promoter sequenceof human neurofilament L (Charron, J. Biol. Chem 270 (1995),25739-25745). Beside elements which are responsible for the initiationof transcription such regulatory elements may also comprisetranscription termination signals, such as SV40-poly-A site or thetk-poly-A site, downstream of the polynucleotide. In this context,suitable expression vectors are known in the art such as Okayama-BergcDNA expression vector pcDV1 Pharmacia), pRc/CMV, pcDNA1, pcDNA3(In-Vitrogene, as used, inter alia in the appended examples), pSPORT1(GIBCO BRL) or pGEMHE Promega). Beside the nucleic acid moleculesdefined herein, the vector may further comprise nucleic acid sequencesencoding for secretion signals. Such sequences are well known to theperson skilled in the art. Furthermore, depending on the expressionsystem used leader sequences capable of directing the protein/(poly)peptide to a cellular compartment may be added to the coding sequence ofthe nucleic acid molecules of the invention and are well known in theart. The leader sequence (s) is (are) assembled in appropriate phasewith translation, initiation and termination sequences, and preferably,a leader sequence capable of directing secretion of translated protein,or a part thereof.

As mentioned herein above, said vector may also be, besides anexpression vector, a gene transfer and/or gene targeting vector. Genetherapy, which is based on introducing therapeutic genes into cells byex-vivo or in-vivo techniques is one of the most important applicationsof gene transfer. Suitable vectors, vector systems and methods forin-vitro or in-vivo gene therapy are described in the literature and areknown to the person skilled in the art; see, e. g., Giordano, NatureMedicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919;Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996),370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, NatureMedicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, Schaper, CurrentOpinion in Biotechnology 7 (1996), 635-640 Verma, Nature 389 (1997),239-242 WO 94/29469, WO 97/00957, U.S. Pat. Nos. 5,580,859, 589,66 or4,394,448 and references cited therein.

In particular, said vectors and/or gene delivery systems are alsodescribed in gene therapy approaches in neurological tissue/cells (see,inter alia Blomer, J. Virology 71 (1997) 6641-6649) or in thehypothalamus (see, inter alia, Geddes, Front Neuroendocrinol. 20 (1999),296-316 or Geddes, Nat. Med. 3 (1997), 1402-1404).

Further suitable gene therapy constructs for use in neurologicalcells/tissues are known in the art, for example in Meier (1999), J.Neuropathol. Exp. Neurol. 58,10991110. The nucleic acid molecules andvectors of the invention may be designed for direct introduction or forintroduction via liposomes, viral vectors (e. g. adenoviral,retroviral), electroporation, ballistic (e. g. gene gun) or otherdelivery systems into the cell. Additionally, a baculoviral system canbe used as eukaryotic expression system for the nucleic acid moleculesdescribed herein.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacological and/or physiologicaleffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of partially or completely curing a disease and/oradverse effect attributed to the disease. The term “treatment” as usedherein covers any treatment of a disease in a mammal, particularly ahuman, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i. e. arresting itsdevelopment; or (c) relieving the disease, i. e. causing regression ofthe disease.

In yet another embodiment, the present invention provides for the use ofa RGM or Neogenin polypeptide or of a functional fragment or derivativethereof or of a polynucleotide encoding said polypeptide or fragment orderivative as a marker of stem cells. Since it is envisaged that stemcells as well as their undifferentiated progenitor cells express RGM andNeogenin, RGM and Neogenin (and/or functional fragments or derivativesthereof) may be employed to influence thedifferentiation/differentiation pattern of said stem cells.

It is furthermore envisaged that antibodies directed against RGM orNeogenin or functional fragment(s)/derivative(s) thereof may be employedto influence the differentiation of (neuronal) stem cells and (neuronal)progenitor cells. It is particularly preferred that said antibodies (aswell as other RGM-inhibitors and/or RGM-binding molecules) be employedto selectively label stem cells. Therefore these reagents may beemployed as markers for stem cells. It is also envisaged that peptidesor derivatives be employed in said purpose.

In a particularly preferred embodiment of the present invention, thepolypeptide and/or fragment thereof which comprises or has an RGM aminoacid sequence to be used in accordance with their invention is asoluble, i. e. not membrane bound molecule.

As shown in Davis (1994), Science 266,816-819, ephrins, in particularA-ephrins, are not active in soluble, monomeric form. In contrast,soluble RGMs are active and may function without anymembrane-attachment. RGM, in contrast to ephrins, is capable ofself-formation of dimers and/or of the formation of higher aggregates.The invention also provides for the use of a RGM molecule or functionalfragment or derivative thereof or of a polynucleotide encoding saidpolypeptide or a fragment or a derivative for the preparation of apharmaceutical composition for alleviating, preventing and/or treatinghomeostatic and/or bleeding disorders and/or vascular damage.

It is envisaged, without being bound by theory, that RGMs may, due totheir structural homology to von-Willebrand factor (vWF), be employed inthe treatment of said disorders/diseases. Furthermore, it is envisagedthat RGM may interact with von Willebrand factor and that said molecule,thereby, influences the activity of vWF.

Furthermore, the inhibitors as defined herein should be employed indisorders where immune cells invade the brain, like multiple sclerosis,encephalomyelitis disseminata.

The present invention also provides for the use of an antibody or afragment or a derivative thereof, or an aptamer, or a binding moleculecapable of interacting with a polypeptide having or comprising the RGMor Neogenin amino acid sequence or with functional fragment orderivative thereof or of a nucleic acid molecule capable of interactingwith a polynucleotide encoding said polypeptide or a fragment thereoffor the preparation of a diagnostic composition for detectingneurological and/or neurodegenerative disorders or dispositions thereto.

The diagnostic composition may be used, inter alia, for methods fordetermining the expression of the nucleic acids encoding RGM andNeogenin polypeptides by detecting, inter alia, the presence of thecorresponding mRNA which comprises isolation of RNA from a cell,contacting the RNA so obtained with a nucleic acid probe as describedabove under hybridizing conditions, and detecting the presence of mRNAshybridized to the probe.

Furthermore, corresponding mutations and/or alterations may be detected.

Furthermore, RGM and Neogenin (poly) peptides can be detected withmethods known in the art, which comprise, inter alia, immunologicalmethods, like, ELISA or Western blotting.

The diagnostic composition of the invention may be useful, inter alia,in detecting the prevalence, the onset or the progress of a diseaserelated to the aberrant expression of a RGM or Neogenin polypeptide.Accordingly, the diagnostic composition of the invention may be used,inter alia, for assessing the prevalence, the onset and/or the diseasestatus of neurological, neurodegenerative and/or inflammatory disorders,as defined herein above. It is also contemplated that anti-RGM oranti-Neogenin antibodies, aptamers etc. and compositions comprising suchantibodies, aptamers, etc. may be useful in discriminating the stage(s)of a disease.

The diagnostic composition optionally comprises suitable means fordetection. The nucleic acid molecule(s), vector(s), antibody(ies),(poly)peptide(s), described above are, for example, suitable for use inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier. Examples of well-known carriers include glass,polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble or insoluble for the purposes of theinvention.

Solid phase carriers are known to those in the art and may comprisepolystyrene beads, latex beads, magnetic beads, colloid metal particles,glass and/or silicon chips and surfaces, nitrocellulose strips,membranes, sheets, duracytes and the walls of wells of a reaction tray,plastic tubes or other test tubes. Suitable methods of immobilizingnucleic acid molecule(s), vector(s), host(s), antibody(ies),(poly)peptide(s), fusion protein(s) etc. on solid phases include but arenot limited to ionic, hydrophobic, covalent interactions and the like.Examples of immunoassays which can utilize said compounds of theinvention are competitive and non-competitive immunoassays in either adirect or indirect format. Commonly used detection assays can compriseradioisotopic or non-radioisotopic methods.

Examples of such immunoassays are the radioimmunoassay (RIA), thesandwich (immunometric assay) and the Northern or Southern blot assay.Furthermore, these detection methods comprise, inter alia, IRMA (ImmuneRadioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (EnzymeLinked Immuno Assay), FIA fluorescent immune Assay), and CLIA(Chemioluminescent Immune Assay).

Furthermore, the diagnostic compounds of the present invention may beare employed in techniques like FRET (Fluorescence Resonance EnergyTransfer) assays.

The nucleic acid sequences encoding RGMs of other species as well asvariants of RGMs are easily deducible from the information providedherein. These nucleic acid sequences are particularly useful, as pointedout herein above, in medical and/or diagnostic setting, but they alsoprovide for important research tools. These tools may be employed, interalia, for the generation of transgenic animals which overexpress orsuppress RGMs or wherein the RGM gene is silenced and/or deleted.Furthermore, said sequences may be employed to detect and/or elucidateRGM interaction partners and/or molecules binding to and/or interferingwith RGMs. The same holds true for nucleic acid sequences encodingNeogenin.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

The following Examples are offered by way of illustration and not by wayof limitation:

EXAMPLES Example 1

An expression vector containing a vector-derived signal sequence, thechick RGM sequence from aa 28 to 403 fused to AP and a Myc His tag wasconstructed. This plasmid was stably transfected into HEK293 cells andsecreted RGM-AP protein was purified on a Ni-containing resin.Quantitative binding assays to transfected COS-7 cells were conducted asdescribed for Nogo-AP binding¹³. To isolate a cDNA encoding a chickRGM-AP binding protein, a mouse adult brain cDNA library (Origene) wasscreened with 10 nM RGM-AP, as described previously for Nogo-66-AP¹³.Mouse RGM-1-AP (Accession number BC023870) and RGM-2-AP (AK080819) wereprepared by identical methods as for chick RGM-AP. Mouse RGM-3 isencoded by BC022603. The mouse Unc5H1 and Unc5H3 expression plasmidswere derived from EST clones (BI818609 and BI769500) and the pCMV-SPORT6vector. Truncated versions of chick Neogenin-1 were expressed using thepcDNA3.1-MycHis vector. The soluble ectodomain protein contains aaresidues 1-1027 of chick Neogenin-1, the ecto+TM protein containsresidues 1-1115 and the 6×FNIII+TM protein contains aa 400-11.15. Arabbit anti-mouse neogenin-1 antibody was emploed for immunoblots (SantaCruz Biotechnology, Inc.).

Chick retinal axons and tectal membranes were prepared for stripe assaysas described^(10,11). Soluble ectodomain of chick Neogenin-1 (1-1027)was expressed with a carboxyl MycHis tag as a secreted protein in HEK293cells and purified on a Ni containing resin. Dialyzed protein was addedto the stripe assay cultures. Recombinant RGM-AP-MycHis and ephrinA2-Fcor ephrinA5-Fc stripes were prepared using an initial coating ofpoly-L-lysine coated coverslips with anti-Myc antibody or anti-Human IgGantibody as described for other proteins^(10,11,14).

Example 2

Ligand Screening of Transfected COS Cells.

I. Prepare the Ligand

Expression Construct: cDNA encoding the targeted RGM is tagged with theFc—portion of human IgG and subcloned into a 293 expression vector(pCEP4: In Vitrogen).

Transfection: 293 EBNA cells are transfected (CaPO.sub.4 method) withthe RGM expression construct. After 24 h recovery, transfected cells areselected with G418 (geneticin, 250 ug/ml, Gibco) and hygromycin (200ug/ml). Once the selection process is complete, cells are maintained inDulbecco's Modified Eagles medium (DME)/10% FCS under selection.

Preparation of Conditioned Medium: Serum-containing media is replacedwith Optimem with glutamax-1 (Gibco) and 300 ng/ml heparin (Sigma), andthe cells are conditioned for 3 days. The media is collected and spun at3,000.times.g for 10 minutes. The supernatant is filtered (0.45 um) andstored with 0.1% azide at 4.degree. C. for no more than 2 weeks.

II. Prepare Truncated Receptor (Positive Control)

Expression Construct: cDNA encoding a corresponding Neogenin deletionmutant comprising the extracellular domain (truncated immediatelyN-terminal to the transmembrane region) is subcloned into a 293expression vector (pCEP4: In Vitrogen).

Transfection: 293 EBNA cells are transfected (CaPO.sub.4 method) withthe receptor mutant expression construct. After 24 h recovery,transfected cells are selected with G418 (geneticin, 250 ug/ml, Gibco)and hygromycin (200 ug/ml). Once the selection process is complete,cells are maintained in Dulbecco's Modified Eagles medium (DME)/10% FCSunder selection.

Preparation of Conditioned Medium: Serum-containing media is replacedwith Optimem with glutamax-1 (Gibco) and 300 ng/ml heparin (Sigma), andthe cells are conditioned for 3 days. The media is collected and spun at3,000.times.g for 10 minutes. The supernatant is filtered (0.45 um) andstored with 0.1% azide at 4.degree. C. for no more than 2 weeks.

III. Transfect COS Cells

Seed COS cells (250,000) on 35 mm dishes in 2 ml DME/10% FCS. 18-24 hlater, dilute 1 ug of netrin receptor-encoding DNA (cDNA cloned intopMT21 expression vector) into 200 ul serum-free media and add 6 ul ofLipofectamine (Gibco). Incubate this solution at room temperature for15-45 min.

Wash the cells 2× with PBS. Add 800 ul serum-free media to the tubecontaining the lipid-DNA complexes. Overlay this solution onto thewashed cells.

Incubate for 6 h. Stop the reaction by adding 1 ml DMA/20% FCS. Refeedcells. Assay cells 12 hr later.

IV. Ligand Binding Assay

Wash plates of transfected COS cells 1× with cold PBS (plus Ca/Mg)/1%goat serum.

Add 1 ml conditioned media neat and incubate 90 min at room temp.

Wash plates 3× with PBS (plus Ca/Mg). On the 4th wash, add 1 ml 50%methanol to 1 ml PBS. Then add 1 ml methanol. Evacuate and add 1 mlmethanol.

Wash 1× with PBS. Wash 1X PBS/1% goat serum.

Add secondary antibody (1-to-2,000 anti-human Fc conjugated to alkalinephosphatase (Jackson Lab)) in PBS/1% goat serum. Incubate 30-40 mlinroom ternp.

Wash 3× with PBS. Wash 1× alkaline phosphatase buffer (100 mM Tris-Cl,pH 9.5, 100 mM NaCl, 5 mM MgCl). Prepare alkaline phosphatase reagents:4.5 ul/ml NBT and 3.5 ul/ml BCIP (Gibco) in alkaline phosphatase buffer.

Incubate 10-30 min, quench with 20 mM EDTA in PBS. Cells that have boundRGM are visible by the presence of a dark purple reaction product.

In parallel incubations, positive controls are provided by titrating RGMbinding with serial dilutions of the mutant receptor conditioned medium.

V. Results: Binding of RGM to Neogenin

Cell expressing mammalian RGM were shown to bind Neogenin. No reactivitywas observed with control COS cells or with receptor-expressing COScells in the presence of the secondary antibody but in the absence ofthe RGM-Fc fusion.

Binding was observed to receptor-expression cells using a construct inwhich RGM is fused directly to alkaline phosphatase, for which asecondary antibody is not required. Neogenin deletion mutants titratethe RGM-receptor binding, serving as a positive control for inhibitionassays.

Example 3

Comparison of axonal guidance phenotypes in Neogenin, DCC, Netrin, andRGM null mice.

In order to assess the functional role of the RGM/Neogenin system inneurological outcome after brain or spinal cord injury, studies in micewith targeted gene deletions are studied. These mice are created usingmouse Embryonic Stem (ES) cells selected to contain disruptions of theendogenous genes of interest. The ES cells with gene disruptions isinjected into mouse blastocysts to derive chimeric animals and then thetargeted mutation are bred to homozygosity. In mice lacking Neogenin orRGM1 or RGM2 or RGM3 functional protein, various mouse models for humanneurological disease are studied. For example, middle cerebral arteryocclusion (MCAO) is created in mice using an intraluminal thread bystandard methods. This MCAO produces a stroke in the brain andfunctional deficits in behavior. The recovery of mice from such injuryin wild type and gene targeted lines is compared. The RGM/Neogenininteraction limits recovery from injury. Parallel studies of braintrauma and spinal cord traums are also made with mice lacking Neogeninor RGM1 or RGM2 or RGM3 function. Brain trauma is created by fluidpercussion and spinal cord injury is created by either transection or bycontusion. Improved recovery of mouse behavior after these traumaticlesions demonstrates the role of the RGM/Neogenin interaction inlimiting recovery from CNS damage. Agents demonstrated to be inhibitoryto the RGM/Neogenin interaction similarly improve recovery in wild-typemice exposed to brain trauma/spinal cord injury etc.

REFERENCES

-   1. Tessier-Lavigne, M. & Goodman, C. S. The molecular biology of    axon guidance. Science 274, 1123-1133. (1996).-   2. Yu, T. W. & Bargmann, C. I. Dynamic regulation of axon guidance.    Nat Neurosci 4 Suppl, 1169-76 (2001).-   3. Monnier, P. P. et al. RGM is a repulsive guidance molecule for    retinal axons. Nature 419, 392-5 (2002).-   4. Wang, H., Copeland, N. G., Gilbert, D. J., Jenkins, N. A. &    Tessier-Lavigne, M. Netrin-3, a mouse homolog of human NTN2L, is    highly expressed in sensory ganglia and shows differential binding    to netrin receptors. J Neurosci 19,4938-47 (1999).-   5. Feldheim, D. A. et al. Genetic analysis of ephrin-A2 and    ephrin-A5 shows their requirement in multiple aspects of    retinocollicular mapping. Neuron 25, 563-74 (2000).-   6. Fazeli, A. et al. Phenotype of mice lacking functional Deleted in    colorectal cancer (Dcc) gene. Nature 386, 796-804 (1997).-   7. Serafini, T. et al. Netrin-1 is required for commissural axon    guidance in the developing vertebrate nervous system. Cell 87,    1001-14 (1996).-   8. Hong, K et al. A ligand-gated association between cytoplasmic    domains of UNC5 and DCC family receptors converts netrin-induced    growth cone attraction to repulsion. Cell 97, 927-41 (1999).-   9. Geisbrecht, B. V., Dowd, K. A., Barfield, R. W., Longo, P. A. &    Leahy, D. J. Netrin binds discrete subdomains of DCC and UNC5 and    mediates interactions between DCC and heparin. J Biol Chem (2003).-   10. Walter, J., Kern-Veits, B., Huf, J., Stolze, B. & Bonhoeffer, F.    Recognition of position-specific properties of tectal cell membranes    by retinal axons in vitro. Development 101, 685-96 (1987).-   11. Walter, J., Henke-Fahle, S. & Bonhoeffer, F. Avoidance of    posterior tectal membranes by temporal retinal axons. Development    101,909-13 (1987).-   12. Ming, G. L. et al. cAMP-dependent growth cone guidance by    netrin-1. Neuron 19, 1225-35 (1997).-   13. Fournier, A. E., GrandPre, T. & Strittmatter, S. M.    Identification of a receptor mediating Nogo-66 inhibition of axonal    regeneration. Nature 409, 341-6 (2001).-   14. Vielmetter, J., Stolze, B., Bonhoeffer, F. & Stuermer, C. A. In    vitro assay to test differential substrate affinities of growing    axons and migratory cells. Exp Brain Res 81,283-7 (1990).

1. A method for identifying an agent which modulates the binding of aRepulsive Guidance Molecule (RGM) to a Neogenin, the method comprisingthe steps of: (a) forming a mixture comprising an isolated mammalian RGMand an isolated mammalian Neogenin, wherein the isolated mammalianNeogenin has the amino acid sequence of SEQ ID NO: 1; (b) incubatingsaid mixture in the presence of an agent; and (c) detecting in theincubated mixture of step (b) the level of specific binding between saidRGM and said Neogenin, wherein a difference in the detected level ofspecific binding of said RGM to said Neogenin in the presence of saidagent relative to the level of specific binding in the absence of saidagent indicates that said agent modulates the binding of said RGM tosaid Neogenin, wherein said RGM is RGM A or RGM B.
 2. A method formonitoring the binding of a Repulsive Guidance Molecule (RGM), whereinsaid RGM is RGM A or RGM B to a Neogenin, the method comprising thesteps of: (a) contacting a first protein comprising said RGM tagged witha visible stain or enzymatic signal, with a second protein whichcomprises the Neogenin, wherein said Neogenin has the amino acidsequence of SEQ ID NO: 1 and with a RGM A-specific antibody, or a RGM Bspecific antibody which will interfere in the binding between the taggedRGM A or RGM B and the Neogenin; (b) leaving the mixture for a time andunder conditions where a domain of the RGM A or RGM B binds to a domainof the Neogenin; and (c) monitoring the binding of the first proteinwhich comprises the tagged RGM, to the second protein which comprisesthe Neogenin, wherein a reduction in the visible stain or enzymaticsignal indicates a reduction of tagged RGM binding to Neogenin due tothe antibody interacting with said binding.